Retinoid suppression of ventricular muscle cell hypertrophy

ABSTRACT

The invention features methods of identifying a compound which suppresses ventricular muscle cell hypertrophy. In one embodiment, ventricular muscle cells are contacted with a test compound, which acts through an RAR or RXR receptor, in the presence of an inducer of hypertrophy. Development of ventricular muscle cell hypertrophy is then measured to identify compounds having the desired activity.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.09/041,050, filed Mar. 10, 1998 now U.S. Pat. No. 6,060,311, which is adivision of 08/685,339 filed Jul. 22, 1996, U.S. Pat. No. 5,767,155,which claims benefit of provisional application Ser. No. 60/019,016,filed Jul. 28, 1995, which application is incorporated herein byreference.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was supported in part by National Cancer Institute grantsCA54418, HD27183, NHLBI grants HL45069, HL53773, HL46345, and 91-022170.The U.S. Government may have rights in this invention.

TECHNICAL FIELD

This invention relates to compounds which affect ventricular muscle cellhypertrophy, an assay for identifying such compounds, and to methods formodulating cardiac function in the treatment of heart disorders.

BACKGROUND OF THE INVENTION

Heart failure affects approximately three million Americans, developingat a rate of approximately 400,000 new cases per year. Current therapyfor heart failure is primarily directed to using angiotensin-convertingenzyme (ACE) inhibitors and diuretics. ACE inhibitors appear to slow theprogression towards end-stage heart failure in patients; however, theyare unable to relieve symptoms in more than 60% of heart failurepatients and reduce mortality of heart failure only by approximately15-20%. Heart transplantation is limited by the availability of donorhearts. With the exception of digoxin, the chronic administration ofpositive inotropic agents has not resulted in a useful drug withoutaccompanying adverse side effects, such as increased arrhythmia, suddendeath, or other deleterious side effects related to survival. Thesedeficiencies in current therapy suggest the need for additionaltherapeutic approaches.

Cardiac muscle hypertrophy is one of the most important adaptivephysiological responses of the myocardium. In response to increaseddemands for cardiac work or following a variety of pathological stimuliwhich lead to cardiac injury, the heart adapts through the activation ofa hypertrophic response in individual cardiac muscle cells, which ischaracterized by an increase in myocyte size, the accumulation ofcontractile proteins within individual cardiac cells, the activation ofembryonic gene markers expression, and the lack of a concomitant effecton muscle cell proliferation. Although the hypertrophic process caninitially be compensatory, there can be a pathological transition inwhich the myocardium becomes dysfunctional (Braunwald (1994) inPathophysiology of Heart Failure, (Braunwald, ed.); Saunders,Philadelphia; Vol. 14, pp 393-402).

Studies in an in vitro model system of ventricular muscle cellhypertrophy have led to the identification of a number of mechanical,hormonal, growth factor, and pathological stimuli which can activateseveral independent features of hypertrophy (Chien et al. (1991) FASEBJ. 5:3037-3046; Knowlton et al. (1991.) J. Biol. Chem. 266:7759-7768;Shubeita et al. (1990) J. Biol. Chem. 265:20555-20562; Thorburn et al.(1993) J. Biol. Chem. 268:2244-2249; LaMorte et al. (1994) J. Biol.Chem. 269:13490-13496; Knowlton et al. (1993) J. Biol. Chem.268:15374-15380). Currently, there are at least two signal transductionpathways, involving both ras- (Thorburn et al. (1993) supra), and G_(q)protein-dependent downstream effectors (LaMorte et al. (1994) supra)implicated in the activation of features of the hypertrophic response inthe in vitro model system.

While progress has been made in uncovering the signaling pathways whichactivate the ventricular muscle cell hypertrophic response, relativelylittle is known as to mechanisms which inhibit or suppress thehypertrophic response.

There is a need for an improved heart failure therapy, such ascongestive heart failure and hypertrophic cardiomyopathy.

SUMMARY OF THE INVENTION

The present invention is based in part on the discovery that retinoicacid suppresses α₁-adrenergic agonist- and endothelin-mediatedventricular muscle cell hypertrophy. Accordingly, the invention featuresa method of suppressing ventricular muscle cell-hypertrophy bycontacting ventricular muscle cells with an effective amount of aretinoic acid compound, retinoic acid derivative, or pharmaceutical saltthereof.

The invention features a method of identifying compounds which suppressventricular muscle cell hypertrophy, comprising contacting ventricularmuscle cells with a test compound in the presence of an inducer ofventricular muscle cell hypertrophy, and measuring the development ofventricular muscle cell hypertrophy.

The development of hypertrophy in ventricular muscle cells is measuredin cells exposed to an inducer of ventricular muscle cell hypertrophywith and without the test compound, and the development of hypertrophycompared. The development of ventricular muscle cell hypertrophy ismeasured in a variety of ways, including by increase in cell size,induction of a genetic marker of ventricular muscle cell hypertrophy,increase in the assembly of an individual contractile protein such asmyosin light chain-2v (MLC-2v) into organized contractile units,accumulation of contractile units, activation of a program of immediateearly gene expression, and the induction of genes encoding contractileand embryonic proteins. In a specific embodiment, ventricular musclecell hypertrophy is measured by determining the expression of an atrialmarker, for example, atrial natriuretic factor (ANF).

In one embodiment of the method of identifying compounds which suppressventricular muscle cell hypertrophy, cells are incubated with an inducerof α₁-adrenergic-mediated hypertrophy with and without the testcompound, and the ability of the test compound to suppress developmentof α₁-adrenergic-mediated ventricular muscle cell hypertrophy measured.In another embodiment, the cells are incubated with an endothelin withand without the test compound, and the ability of the test compound tosuppress endothelin-mediated ventricular muscle cell hypertrophydetermined. A test compound which suppresses ventricular muscle cellhypertrophy may block a hypertrophic pathway in a variety of ways,including by activating retinoic acid-specific receptors, e.g., RAR.

The invention features a method for identifying compounds which inhibitretinoic acid suppression of ventricular muscle cell hypertrophy. Acompound may inhibit retinoic acid suppression of ventricular musclecell hypertrophy by blocking, suppressing, reversing, or antagonizingthe action of the retinoic acid action. In one embodiment, cells areincubated with an inducer of ventricular muscle cell hypertrophy in thepresence of a retinoic acid compound that suppresses the development ofventricular muscle cell hypertrophy, and with and without the testcompound, and the ability of the test compound to block the retinoicacid compound suppression of ventricular muscle cell hypertrophy ismeasured. The inducer of ventricular muscle cell hypertrophy may be anα₁-adrenergic agonist or endothelin.

The invention features a therapeutic method for treatment or preventionof ventricular muscle cell hypertrophy-mediated heart failure in amammal comprising administering an therapeutically effective amount of aretinoic acid compound. In one embodiment, the therapeutic method oftreating heart failure includes treating or preventingα₁-adrenergic-mediated ventricular muscle cell hypertrophy. In anotherembodiment, the therapeutic method treats or preventsendothelin-mediated ventricular muscle cell hypertrophy.

Other aspects of the invention will become apparent from the followingdetailed description and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graph showing the effect of Phe and RA on induction of geneexpression in cells transfected with an ANF promoter-luciferase fusiongene construct. Transfected cells were cultured in maintenance medium(1:-RA-Phe), 2×10⁻⁶ M Phe (2:-RA+Phe), or 1×10⁻⁶ M RA+2×10⁻⁶ M Phe(3:+RA+Phe). Bars represent the effects of these agents relative tounstimulated cells. The luciferase activities were normalized by β-galactivities through co-transfection with a CMV-β-gal plasmid.

FIGS. 1B is a graph showing the effect of Phe and RA on induction ofgene expression in cells transfected with an Rous sarcoma virus (RSV)promoter-luciferase fusion gene construct. Transfected cells werecultured in maintenance medium (1:-RA-Phe), 2×10⁻⁶ M Phe (2:-RA+Phe), or1×10⁻⁶ M RA+2×10⁻⁶ M Phe (3:+RA+Phe). Bars represent the effects ofthese agents relative to unstimulated cells. The luciferase activitieswere normalized by β-gal activities through co-transfection with aCMV-β-gal plasmid.

FIG. 1C is a graph showing the induction of luciferase expression incells transfected with a ANF promoter-luciferase fusion gene construct.Transfected cells were cultured in maintenance medium, 2×10⁻⁶ M Phe,1×10⁻⁸ M endothelin, or 10% FBS, with or without 1×10⁻⁶ M RA. Barsrepresent the effects of these agents relative to unstimulated cells.The luciferase activities were normalized by β-gal activities throughco-transfection a CMV-β-gal plasmid.

FIG. 2A is a graph showing activation of RA receptors in culturedventricular cells. Reporter plasmids containing CRBP II-luciferase orβ-RE I-luciferase were transfected into ventricular muscle cells withoutor with co-transfected expression plasmids containing human RXRα or RARαCDNA driven by a CMV promoter. 9-cis RA or all-trans RA were separatelyadded to the cultures to activate CRBP II or β-RE I promoters. Barsrepresent the effects of ligands on the promoter activities relative toreporter gene activity in the absence of ligand.

FIG. 2B is a graph showing the effect of Phe+RA on activation of areporter construct containing the-ANF promoter alone; the ANF promoterwith a dominant negative reporter construct (CMV-hRAR403), the wild-typereceptor construct (CMV-hRXR), or the wild-type receptor constructCMV-HRAR in ventricular muscle cells cultured in maintenance medium, or2×10⁻⁶ M Phe with or without 1×10⁻⁷ M all-trans RA. The percent of Pheinduction is expressed as the ratio of fold increase in the reportergene by Phe in the presence of RA to the fold increase in the reportergene observed following Phe stimulation in the absence of RA.

FIG. 3A is a graph of induction of luciferase expression in cellstransfected with the ANF-luciferase reporter gene construct andactivated by 2×10⁻⁶ M Phe with a series of concentrations of threeagonists; LG64,(E)-4-[2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthalenyl)-1-propenyl]benzoicacid (TTNPB), and(E)-4-[2-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthalenyl)-1-propenyl]benzoicacid (3-methyl-TTNBP). Curves represent the relative luciferaseactivities normalized by the β-gal activities.

FIG. 3B is a graph of the induction of luciferase activity in cellstransfected with the ANF-luciferase reporter gene construct and exposedto 2×10⁻⁷ M Phe and one of the following: 1×10⁻⁷ M dexamethasone (Dex),1×10⁻⁷ M thyroid hormone (T3), 1×10⁻⁷ M estrogen (E2), 1×10⁻⁷ M vitaminD (D3), 1×10⁻⁷ M linoleic acid (LA), and 1×10⁻⁷ M retinoic acid (RA).

DETAILED DESCRIPTION

Utilizing an in vitro system of ventricular muscle cell hypertrophy, arole for retinoic acid compounds in suppressing the activation of thehypertrophic response by two well-defined hormonal stimuli, α-adrenergicagonists and endothelin-1, is demonstrated herein. The present inventiondemonstrates that hypertrophy suppressor pathways exist within cardiacmuscle cells, and that retinoic acid compounds can activate thesepathways. Thus, the invention provides a useful method for the treatmentand prevention of ventricular muscle cell hypertrophy and foridentifying compounds which activate hypertrophy suppressor pathways.

Before the methods of the invention are described, it is to beunderstood that this invention is not limited to the particular methodsdescribed. The terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting sincethe scope of the present invention will be limited only by the appendedclaims.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural references unless the contextclearly dictates otherwise. Thus, for example, references to “retinoidacid” or “a retinoic acid compound” include mixtures of such retinoicacids, retinoids, and/or retinoic acid-like compounds, reference to “theformulation” or “the method” includes one or more formulations, methods,and/or steps of the type described herein and/or which will becomeapparent to those persons skilled in the art upon reading thisdisclosure and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated herein by reference for the purpose of disclosing anddescribing the material for which the reference was cited in connectionwith.

Definitions

“Ventricular muscle cell hypertrophy” is a condition A characterized byan increase in the size of individual ventricular muscle cells, theincrease in cell size being sufficient to result in a clinical diagnosisof the patient or sufficient as to allow the cells to be determined aslarger (e.g., 2-fold or more larger than non-hypertrophic cells). It maybe accompanied by accumulation of contractile proteins within theindividual cardiac cells and activation of embryonic gene expression.

In vitro and in vivo methods for determining the presence of ventricularmuscle cell hypertrophy are known. In vitro assays for ventricularmuscle cell hypertrophy include those methods described herein, e.g.,increased cell size and increased expression on atrial natriureticfactor (ANF). Changes in cell size are used in a scoring system todetermine the extent of hypertrophy. These changes can be viewed with aninverted phase microscope, and the degree of hypertrophy scored with anarbitrary scale of 7 to 0, with 7 being fully hypertrophied cells, and 3being non-stimulated cells. The 3 and 7 states may be seen in Simpson etal., (1982) Circulation Res. 51:787-801, FIG. 2, A and B. respectively.The correlation between hypertrophy score and cell surface area (μm²)has been determined to be linear (correlation coefficient =0.99) (Dr.Kathleen L. King, Genentech, Calif.). In phenylephrine-inducedhypertrophy, non-exposed (normal) cells have a hypertrophy score of 3and a surface area/cell of 581 μm and fully hypertrophied cells have ahypertrophy score of 7 and a surface area/cell of 1811 μm², orapproximately 200% of normal. Cells with a hypertrophy score of 4 have asurface area/cell of 771 AiO, or approximately 30% greater size thannon-exposed cells; cells with a hypertrophy score of 5 have a surfacearea/cell of 1109 amid or approximately 90% greater size thannon-exposed cells; and cells with a hypertrophy score of 6 have asurface area/cell of 1366 μm², or approximately 135% greater size thannon-exposed cells. The presence of ventricular muscle cell hypertrophypreferably includes cells exhibiting an increased size of about 15%(hypertrophy score 3.5) or more. Inducers of hypertrophy vary in theirability to induce a maximal hypertrophic response as scored by theabove-described assay. For example, the maximal increase in cell sizeinduced by endothelin is approximately a hypertrophy score of 5.

“Suppression” of ventricular muscle cell hypertrophy means a reductionin one of the parameters indicating hypertrophy relative to thehypertrophic condition, or a prevention of an increase in one of theparameters indicating hypertrophy relative to the normal condition. Forexample, suppression of ventricular muscle cell hypertrophy can bemeasured as a reduction in cell size relative to the hypertrophiccondition. Suppression of ventricular muscle cell hypertrophy means adecrease of cell size of 10% or greater relative to that observed in thehypertrophic condition. More preferably, suppression of hypertrophymeans a decrease of cell size of 30% or greater; most preferably,suppression of hypertrophy means a decrease of cell size of 50% or more.Relative to the hypertrophy score assay when phenylephrine is used asthe inducing agent, these decreases would correlate with hypertrophyscores of about 6.5 or less, 5.0-5.5, and 4.0-5.0, respectively. When adifferent agent is used as the inducing agent, suppression is measuredrelative to the maximum cell size (or hypertrophic score) measured inthe presence of that inducer.

Prevention of ventricular muscle cell hypertrophy is determined bypreventing an increase in cell size relative to normal cells, in thepresence of a concentration of inducer sufficient to fully inducehypertrophy. For example, prevention of hypertrophy means a cell sizeincrease less than 200% greater than non-induced cells in the presenceof a maximally-stimulating concentration of inducer. More preferably,prevention of hypertrophy means a cell size increase less than 135%greater than non-induced cells; and most preferably, prevention means acell size increase less than 90% greater than non-induced cells.Relative to the hypertrophy score assay when phenylephrine is used asthe inducing agent, prevention of hypertrophy in the presence of amaximally-stimulating concentration of phenylephrine means ahypertrophic score of about 6.0-6.5, 5.0-5.5, and 4.0-4.5, respectively.

In vivo determination of hypertrophy include measurement ofcardiovascular parameters such as blood pressure, heart rate, systemicvascular resistance, contractility, force of heart beat, concentric ordilated hypertrophy, left ventricular systolic pressure, leftventricular mean pressure, left ventricular end-diastolic pressure,cardiac output, stroke index, histological parameters, and ventricularsize and wall thickness. Animal models available for determination ofdevelopment and suppression of ventricular muscle cell hypertrophy invivo include the pressure-overload mouse model, RV murine dysfunctionalmodel, transgenic mouse model, and post-myocardial infarction rat model.Medical methods for assessing the presence, development, and suppressionof ventricular muscle cell hypertrophy in human patients are known, andinclude, for example, measurements of diastolic and systolic parameters,estimates of ventricular mass, and pulmonary vein flows.

The term “retinoic acid” as used herein is intended to include compoundshaving the following structure,

or pharmaceutical salts thereof, derivatives having a structure similarto that of retinoic acid, as well as biologically-equivalent derivativesthereof. Retinoic acid derivatives include natural and syntheticcompounds having biochemically equivalent moieties bound to thecarboxylic carbon atom. Typical salts are the alkalimetal and ammoniumsalts. Particularly preferred salts of the acid includ sodium,potassium, triethanloammonium and ammonium salts.

Combinations of all the foregoing may be used in the method of theinvention. Further, the terms “retinoic acid” are intended to includehydrogenated and nonhydrogenated isomers such as 9-cis-retinol,didehydroretinol, 13-cis-retinoic acid, 13-trans-retinoic acid,all-trans retinoic acid, and didehydroretinoic acid. The retinoic acid,derivatives, and retinoic acid-like molecules useful in the method ofthe invention are those characterized by the ability to bind retinoicacid receptors, thereby initiating the biological sequence of eventsleading to suppression of ventricular muscle cell hypertrophy. Thederivatives may have such a structure when administered or may beconverted to such a structure after administration, i.e., includespro-drugs.

Retinoids are important therapeutic agents in the treatment of cancerand proliferative diseases of the skin (Jong et al. (1993) J. Med. Chem.36:2605-2613). Retinoic acid (RA) and its synthetic analogs (retinoids)effect a wide array of biological processes by activating two distinctclasses of nuclear receptor proteins, the retinoic acid receptors (RARs)(Giguere et al. (1990) Mol. Cell.

Biol. 10:2335-2340) and the retinoid X receptors (RXRS) (Mangelsdorf etal. (1992) Genes Dev. 6:329-344). These retinoid receptors belong to thesteroid/thyroid hormone receptor superfamily (Evans (1988) Science240:889-895), and each class has three receptor subtypes, RAR-α, RAR-β,and RAR-γ, and RXR-α, RXR-β, and RXR-γ. A functional receptor, whereinfunctionality is defined in terms of the ability to activate DNAtranscription, is a dimer, either a homodimer (RXR/RXR) or a heterodimer(RAR/RXR). Retinoic acid 30 derivatives and retinoic acid-like compoundsthat are capable of binding RXR, RAR, or both, include 9-cis-retinoicacid, all-trans retinoic acid,(E)-4-[2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthalenyl)-1-propenyl]benzoicacid (TTNPB), and(E)-4-[2-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthalenyl)-1-propenyl]benzoicacid (3-methyl-TTNPB), and LG64. Evidence suggests a central role forRXR in regulating several distinct hormonal response pathways. Onefunction of RXR is to act as an auxiliary receptor for several nuclearreceptors, including the RARs, thyroid hormone receptors and vitamin Dreceptor. Heterodimers of RXR with these receptors form in solution(Zhang et al. (1992a) Nature 355:4417446) and bind selectively with highaffinity to specific hormone response elements (Hermann et al. (1992)Mol. Endocrinol. 6:1153-1162). RXRs also function independently ashomodimers (Zhang et al. (1992b) Nature 358:587-591) which form in thepresence of the 9-cis isomer of all-trans RA, and have differentresponse element specificities than the RAR:RXR heterodimers (Hermann etal. (1992) supra; Zhang et al. (1992b) supra). In contrast, RARs bindboth ligands with high affinity (Heyman et al. (1992) Cell 68:397-406).The use of molecules which are biologically equivalent, e.g., able tobind retinoic acid receptors to suppress ventricular muscle cellhypertrophy, is included in the method of the invention.

By the term “effective amount” or “therapeutically effective amount” ofretinoic acid is meant an amount of a retinoic acid compound, retinoicacid derivative and/or a retinoic acid-like compound sufficient toobtain the desired physiological effect, e.g., suppression ofventricular muscle cell hypertrophy. An effective amount of retinoicacid is determined by the caregiver in each case on the basis of factorsnormally considered by one skilled in the art to determine appropriatedosages, including the age, sex, and weight of the subject to betreated, the condition being treated, and the severity of the medicalcondition being treated.

The terms “treatment”, “treating” and the like are used herein togenerally mean obtaining a desired pharmacologic and/or physiologiceffect. The effect may by prophylactic in terms of completely orpartially preventing a disease or symptom thereof and/or may betherapeutic in terms of a partial or complete cure for a disease and/oradverse effect attributable to the disease. “Treatment” as used hereincovers any treatment of a disease in a mammal, particularly a human, andincludes:

(a) preventing the disease from occurring in a subject which may bepredisposed to the disease but has not yet been diagnosed as having it;

(b) inhibiting the disease, i.e., arresting its development; or

(c) relieving the disease, i.e., causing regression of the disease. Theinvention is directed to treating patients with or at risk fordevelopment of ventricular muscle cell hypertrophy and relatedconditions mediated by ventricular muscle cell hypertrophy, e.g., heartfailure. More specifically, “treatment ” is intended to mean providing atherapeutically detectable and beneficial effect on a patient sufferingfrom ventricular muscle cell hypertrophy or a condition mediated byventricular muscle cell hypertrophy.

Still more specifically, “treatment” shall mean preventing, alleviating,and/or inhibiting (1) ventricular muscle cell hypertrophy mediatedthrough α₁-adrenergic agonists, (2) ventricular muscle cell hypertrophymediated through endothelin, (3) ventricular muscle cell hypertrophymediated through drugs known to have the adverse effect of promotingcardiac hypertrophy, (4) a medical condition, e.g., heart failure,mediated by ventricular muscle cell hypertrophy induced by α₁-adrenergicagonists, endothelin, and/or drugs which promote cardiac hypertrophy,and (5) ventricular muscle cell hypertrophy initiated by cardiac injury,such as viral myocarditis, long-standing hypertension, cardiomyopathydue to pathological stimuli, and post-myocardial infarction.

By the term “α₁-adrenergic-mediated ventricular muscle cell hypertrophy”is meant ventricular muscle cell hypertrophy which results fromactivation of α₁-adrenergic receptors by α₁-adrenergic agonists.α₁-adrenergic agonists include natural agonists, such as epinephrine,and synthetic agonists, such as phenylephrine or methoxamine.

By the term “endothelin” is meant a peptide compound of the endothelinfamily, including, endothelin-1, endothelin-2, and endothelin-3, whichinduces ventricular muscle cell hypertrophy.

By the term “heart failure” is meant an abnormality of cardiac functionwhere the heart does not pump blood at the rate needed for therequirements of metabolizing tissues. Heart failure includes a widerange of disease states such as congestive heart failure, myocardialinfarction, tachyarrhythmia, familial hypertrophic cardiomyopathy,ischemic heart disease, idiopathic dilated cardiomyopathy, andmyocarditis. The heart failure can be caused by any number of factors,including ischemic, congenital, rheumatic, or idiopathic forms. Chroniccardiac hypertrophy is a significantly diseased state which is aprecursor to congestive heart failure and cardiac arrest. “Treatment”refers to both therapeutic treatment and prophylactic or preventativemeasures, wherein the object is to prevent or slow down (lessen)hypertrophy. Those in need of treatment include those already with thedisorder as well as those prone to have the disorder or those in whichthe disorder is to be prevented. The hypertrophy may be from any causewhich is responsive to retinoic acid, including congenital, viral,idiopathic, cardiotrophic, or myotrophic causes, or as a result ofischemia or ischemic insults such as myocardial infarction. Typically,the treatment is performed to stop or slow the progression ofhypertrophy, especially after heart damage, such as from ischemia, hasoccurred. Preferably, for treatment of myocardial infarctions, theagent(s) is given immediately after the myocardial infarction, toprevent or lessen hypertrophy.

The terms “synergistic”, “synergistic effect” and like are used hereinto describe improved treatment effects obtained by combining one or moretherapeutic agents with one or more retinoic acid compounds. Although asynergistic effect in some field is meant an effect which is more thanadditive (e.g., 1+1=3), in the field of medical therapy an additive(1+1=2) or less than additive (1+1=1.6) effect may be synergistic. Forexample, if each of two drugs were to inhibit the development ofventricular muscle cell hypertrophy by 50% if given individually, itwould not be expected that the two drugs would be combined to completelystop the development of ventricular muscle cell hypertrophy. In manyinstances, due to unacceptable side effects, the two drugs cannot beadministered together. In other instances, the drugs counteract eachother and slow the development of ventricular muscle cell hypertrophy byless than 50% when administered together. Thus, a synergistic effect issaid to be obtained if the two drugs slow the development of ventricularmuscle cell hypertrophy by more than 50% while not causing anunacceptable increase in adverse side effects. “Chronic” administrationrefers to administration of a retinoic acid compound in a continuousmode as opposed to an acute mode, so as to maintain the initialanti-hypertrophic effect for an extended period of time.

Retinoic Acid Supression of Ventricular Muscle Cell Hypertrophy.

To evaluate the effect of a retinoic acid compound on the hypertrophicresponse, an in vitro model system was used in which defined agonistscan activate several independent features of ventricular muscle cellhypertrophy (Example 1). Treatment with the α₁-adrenergic agonistphenylephrine (Phe) results in an increase in ventricular muscle cellsize and assembly of MLC-2v protein into organized sarcomeric unitsrelative to control cells. Phe-stimulated increase in cell size islargely prevented by RA, while myofibrillar organization appears to beintact, and the cells continue to exhibit spontaneous contractility(data not shown). Treatment with RA alone had little effect on theventricular muscle cell phenotype, indicating that the effect of RA wasnot secondary to a toxic cellular effect. Features of hypertrophy arealso induced by the addition of serum. However, the addition of RA hadlittle effect on the serum-treated cells, suggesting that RA has aselective specificity for inhibition of the α₁-adrenergic pathway. Adirect measurement of cell size (Table 1) indicates that Phe or serumincrease cell size approximately 3-fold, while RA treatment specificallysuppresses the Phe induction to near background levels. Taken together,these data indicate that pharmacological concentrations of retinoic acidselectively suppress the hypertrophic response following α₁-adrenergicstimulation.

To examine the effects of RA on the induction of atrial natriureticfactor (ANF), total RNA was isolated from ventricular muscle cellscultured in maintenance media alone, or maintenance media supplementedwith Phe, RA, or both agents (Example 2). Phe induces ANF MRNAexpression by approximately 6-fold relative to control (Table 2). Incontrast, combined treatment with both RA and Phe results in levels ofANF mRNA comparable to unstimulated cells, consistent with an effect ofRA on inhibiting the hypertrophic phenotype. RA suppression of Pheinduction of the ANF mRNA was dose-dependent (Table 3) with a 50%suppression seen with a RA concentration less than 10⁻⁸ M, which iswithin physiological levels and comparable to the dose response profilefor transcriptional activation mediated by retinoid receptors (Gigvereet al. (1987) Nature 330:634-629).

The effects of Phe and RA treatment on the expression of troponin I(TnI) mRNA and myosin light chain 2v (MLC-2v) mRNA was determined, bothconstitutively expressed cardiac muscle genes. As displayed in Table 2,RA had no detectable effect on inhibiting the expression of the TnI orMLC-2v gene during Phe stimulation. Similar results were obtained at theprotein level in studies employing dual immunofluorescence with MLC-2vand ANF antibodies (data not shown). Taken together, these resultsindicate that RA treatment selectively inhibits the expression of agenetic marker of the ventricular muscle cell hypertrophic response,ANF, at both the protein and RNA levels.

Endothelin, a naturally occurring peptide derived from endothelialcells, is a potent constrictor of vascular smooth muscle (Yanagisawa etal. (1989) Trends Pharmacol. Sci. 10:374-378). To date, threeendothelin-related peptides have been identified, endothelin-1, -2, and-3 (Inoue et al. (1989) Proc. Natl. Acad. Sci. USA 86:2863-2867).Endothelin-1 is a 21 amino acid peptide which is a potent venous andarterial vasoconstrictor. The mature biologically active peptide is aproteolytic product of the 38-39 amino acid molecule “big endothelin”(Yanagisawa et al. (1989) supra). Endothelin has been shown to induceprotein tyrosine phosphorylation in aortic smooth muscle cells,mesangial cells, and osteoblast-like cells (Battistini et al. (1993)Peptides 14:385-399), but in neonatal rat ventricular muscle cells inculture, endothelin, like the α-adrenergic agents, stimulatesphosphoinositide hydrolysis and the accumulation of diacylglycerol(Shubeita et al. (1990) supra). Endothelin is present in vivo in bothatrial and ventricular myocardium in healthy and failing hearts andenhances myocardial inotropic activity, vascular smooth muscleproliferation and coronary vasoconstriction (Wei et al. (1994)Circulation 89:1580-1586). Endothelin stimulates multiplecell-signalling pathways in cultured adult cardiac myocytes(Hilal-Dandan et al. (1994) Mol. Pharm. 45:1183-1190; Jones et al.(1992) Am. J. Physiol. 263:H1447-H1454). Several investigators haveshown that endothelin-1 induces hypertrophy of ventricular muscle cellsin vitro (Shubeita et al. (1990) supra; Ito et al. (1991) Circ. Res.69:209-215; Suzuki et al. (1991) J. Cardiovasc. Pharmacol. 17 Suppl.7:S182-S186. See, also U.S. Pat. No. 5,344,644. Endothelin receptorantagonists include compounds such as BQ-123 (Ihara et al. (1992) LifeScience 50:247-0250; Webb et al. (1992) Biochem. Biophys. Res. Commun.185:887-892). BQ-123 is a cyclic pentapeptide that is a potent andspecific blocker of endothelin A receptors, able to block thehypertrophic activity induced by endothelin-1, but not that induced byphenylephrine.

A series of studies have identified cis sequences within the ANFpromoter region which can confer α-adrenergic inducibility to aluciferase reporter gene in transient transfection assays (Knowlton etal. (1991) supra; Argentin et al. (1991) J. Biol. Chem. 266:23315-23322;Grepin et al. (1994) Mol. Cell. Biol. 14:3115-3129). When introducedinto ventricular muscle cells cultured in maintenance media, Phe,endothelin-1, and serum induces a 15- to 20-fold induction of a 3003 ANFpromoter-luciferase fusion gene (Example 3). The induction by Phe orendothelin-1 is suppressed to basal levels by the addition of RA (FIGS.1A and 1C). As a control, the expression of the Rous sarcoma virus (RSV)LTR promoter was not inhibited by RA, and was in fact stimulatedslightly by both Phe and RA (FIG. 1B), indicating the specificity of thesuppressing activity of RA for the ANF promoter.

The induction of the ANF promoter caused by the peptide growth factorendothelin I (FIG. 1C), acts through a specific cell-surface receptorcoupled to signaling pathways which intersect with α-adrenergic signaltransduction, and is thus suppressible by RA. In contrast, RA had littleeffect on suppressing the induction of the ANF luciferase reporter genefollowing serum stimulation, consistent with the results described aboveof the RA effect on serum stimulation of cell size (Example 1). Theinability of RA to suppress serum inducibility of the ANF-luciferasereporter gene suggests that retinoid signaling pathways do not directlyinhibit ANF promoter activity, but rather may induce a functionalblockade at a step in α-adrenergic and endothelin-1 signal transductionpathways of ventricular muscle cell hypertrophy.

To assay the activity of retinoic acid receptors in cultured ventricularmuscle cells, response elements specific for activation by the RXRhomodimer (CRBP II) (Mangelsdorf et al. (1991) supra) or by the RAR/RXRheterodimer (β-RE I) (Sucov et al. (1990) supra) were cloned into anenhancer-dependent heterologous reporter context and introduced bytransfection (Example 4). As shown in FIG. 2A, the CRBP II reporterconstruct was not activated by endogenous receptors in the presence of9cRA, yet was up-regulated when co-transfected with an RXR-expressionconstruct. In contrast, the β-RE I heterodimer reporter gene isfunctional in the presence of ligand using the endogenous complement ofreceptors, and this activity can be further increased by co-transfectionof receptor expression constructs. These results demonstrate thatcultured neonatal ventricular cardiomyocytes express a functionalretinoic acid receptor complex of both RARs and RXRS, which cantransactivate through at least one of the known RA response pathways.

Recently, a truncation deletion mutant of the RARα receptor has beendescribed which functions as a transdominant negative, hRXRα403, able toblock normal retinoic acid induced transcriptional transactivationthrough both the RXR/RAR heterodimer and the RXR homodimer pathways(Damm et al. (1993) Proc. Natl. Acad. Sci. USA 90:2989-2993). Theeffects of expression of this mutant receptor on induction of the ANFpromoter was studied (Example 5) and the results are shown in FIG. 2B.RA was ineffective in suppressing Phe induction of gene expression inthe presence of the dominant negative mutant receptor. Co-transfectionof hRXRα403 with the ANF-luciferase reporter gene in the presence of RAretains 83% of the Phe induction of ANF promoter activity, in comparisonwith only 23% of the Phe inducible activity observed in the absence ofhRXRα403. In contrast, forced expression of either RAR or RXR results inan enhancement of the suppressing activity of RA. These resultsdemonstrate that the suppression of Phe-induced ANF expression by RA ismediated through the endogenous RA receptors.

In cultured cells, atRA and 9cRA are reversibly isomerized;consequently, in transactivation assays, treatment with either compoundpotentially activates both the RXR/RAR heterodimer and RXR homodimerpathways. Recent advances have led to the development of stablesynthetic retinoids which are selective agonists for either the RARs orthe RXRs and which are not subject to metabolic conversion. Among suchagonists, the compound(E)-4-[2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthalenyl)-1-propenyl]benzoicacid (TTNPB) is selective for the RARs, the compound(E)-4-[2-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthalenyl)-1-propenyl]benzoicacid (3-methyl-TTNPB) is a pan-agonist for both the RARs and the RXRS,and the compound LG64 is selective for the RXRS, although there is somedegree of crossover at higher concentrations for both TTNPB and LG64.The ability of these compounds to block Phe induced expression of theANF promoter was tested (Example 5), and the results are shown in FIG.3A. TTNPB and 3-methyl-TTNPB are equivalent in their potency to blockinduction of the ANF promoter, whereas the RXR-selective compound LG64is approximately 40-fold less potent in this assay. Similarly, thehypertrophic increase in cell size induced by Phe was prevented by TTNPBand not by LG64 (data not shown). This indicates that ligand activationof the RAR is the essential component in blocking the α-adrenergicinduction of hypertrophy, as monitored both by cell size and by theexpression of the ANF promoter.

Retinoic acid receptors belong to the larger family of nuclear receptorswhich also mediate the effects of steroid and thyroid hormones andvitamin D. The possibility that these other hormonal signaling pathwaysimpact PE-induced hypertrophy was determined (Example 7). Culturedventricular muscle cells were transfected with the ANF promoter reporterconstruct and co-cultured with PE and additional hormones. As shown inFIG. 3B, only treatment with RA prevented the induction of the ANFpromoter. Treatment with thyroid hormone, estrogen, and vitamin D wereinactive in this assay, and dexamethasone (a synthetic glucocorticoid)actually increased ANF expression. This latter observation has beenpreviously reported and occurs through a non-hypertrophic pathwaywithout an increase in cell size. These results show that theα-adrenergic induction of hypertrophy is specifically blocked by RA, andnot by other hormones.

The specific signal transduction pathways which ultimately result ininduction of ventricular muscle cell hypertrophy are currently unknown.Since retinoic acid is selective in blocking both the α-adrenergic andendothelin-1 pathways of in vitro ventricular muscle cell hypertrophy,but is ineffective on the serum-induced hypertrophic response, it isclear that the ventricular muscle cell is able to modulate its growthresponse through multiple parallel pathways. Furthermore, while RAblocks the increase in cell size and the induction of ANF expressionfollowing α-adrenergic stimulation, RA does not prevent myofibrillarreorganization or the induction of contractility. This suggests thatmultiple effector pathways emerge from the α-adrenergic receptor, as hasin fact been previously documented (Thorburn et al. (1993) supra;LaMorte et al. (1994) supra), and that the inhibitory effects of RA aremanifest at an intermediate point in a portion of the downstreamα-adrenergic pathways, but does not block all of the α-adrenergicreceptor dependent signals.

In addition to directly regulating gene expression, an additionalpathway in which retinoids have biological consequences has beendescribed, in which ligand-activated receptors block the activity of thetranscription factor AP-1 (Schule et al. (1991) Proc. Natl. Acad. Sci.USA 88:6092-6096; Salbert et al. (1993) Molec. Endocrinol. 7:1347-1356).This process, termed cross-coupling, does not require DNA binding of thereceptor, but rather appears to be a protein-protein interaction betweena retinoid receptor and c-jun, and likely additional proteins as well.AP-1 activity is induced in many signal transduction pathways, and theANF promoter has sequences which are similar to AP-1 binding sites(Knowlton et al. (1991) suDra). However, cross-coupling is probably notsufficient to explain at a mechanistic level the-suppression ofhypertrophy caused by retinoic acid. While multiple hormone receptorscan engage in cross-coupling, including the glucocorticoid receptor, theresults presented herein indicate that suppression of hypertrophy is notseen following the addition of glucocorticoids (data not shown).Consequently, the most likely explanation for the observed results isthat retinoic acid induces the expression of a select subset of cardiacgenes through the RXR/RAR heterodimer, and that these induced geneproducts are able to block specific aspects of downstream signaltransduction stimulated by the α-adrenergic and endothelin receptors,leading to suppression of the hypertrophic phenotype.

Retinoic acid has additional consequences on cardiomyocytes in additionto simply suppressing α-adrenergic receptor mediated hypertrophy. Inneonatal cells, RA induces expression of MLC-2v, as shown herein, andhas been previously reported to induce the adult form of α-myosin heavychain (Rohrer et al. (1991) J. Biol. Chem. 266:8638-8646). During thehypertrophic response, ventricular muscle cells express embryonicmarkers, such as the ANF, skeletal α-actin and β-myosin heavy chaingenes, and thereby exit from the mature, adult ventricular phenotype(for review see, Chien et al. (1991) FASEB J. 5:3037-3046). The abilityof retinoids to suppress the expression of atrial and embryonic markersduring hypertrophy in the post-natal state, coupled with their abilityto promote the ventricular phenotype in the embryonic state, raises thepossibility that retinoid dependent pathways function in the adultmyocardium to actively maintain the ventricular phenotype. In thismanner, the activation of a hypertrophic response may be related, inpart, to the relief of retinoid suppression.

The recent demonstration that ras is sufficient to activate hypertrophyin both in vitro and in vivo model systems of hypertrophy is consistentwith the findings herein that retinoic acid compounds suppress thehypertrophic response. In other cell types, retinoic acid compounds caninhibit ras dependent pathways for proliferation and differentiation(Cox et al. (1991) J. Cancer Res. Clin. Oncol. 117:102-108; Leder et al.(1990) Proc. Natl. Acad. Sci. USA 87:9178-9182). In addition, recentstudies in RXRα gene targeted mice have documented a requirement forvitamin A for normal maturation of ventricular muscle cells duringcardiogenesis (Sucov et al. (1994) Genes Dev. 8:1007-1018).RXRα-/-embryos display the persistent, aberrant expression of an atrialmarker (MLC-2a) in the ventricular chamber, a phenotype that is closelyassociated with an embryonic form of heart failure (Dyson et al. (1995)Proc. Natl. Acad. Sci. USA (In Press)). The clear role of vitaminA-dependent pathways in promoting the ventricular phenotype in theembryonic heart is consistent with the findings herein that retinoicacid compounds are also important in maintaining the normal ventricularphenotype in the postnatal state, as well. Since hypertrophy isassociated with the expression of atrial markers in the ventricularchamber (such as ANF), the ability of retinoic acid compounds tomaintain a normal ventricular phenotype in the presence of definedhypertrophic stimuli demonstrates the role of retinoic acid compounds inthe maintenance of the ventricular muscle phenotype in the postnatalstate.

Therapeutic Use

The present invention provides methods for treating or preventing heartfailure and ventricular muscle cell hypertrophy in a mammal by providingan effective amount of a retinoic acid compound. Preferably, the mammalis a human patient suffering from or at risk of developing heartfailure.

The present invention is useful in preventing ventricular muscle cellhypertrophy in patients being treated with a drug which cause cardiachypertrophy, e.g., fludrocortisone acetate. In the method of theinvention, a retinoic acid compound can be given prior to,simultaneously with, or subsequent to a drug which causes cardiachypertrophy.

In the therapeutic method of the invention, a retinoic acid compound isadministered to a human patient chronically or acutely. Optionally,retinoic acid is administered chronically in combination with aneffective amount of a compound that acts to suppress a differenthypertrophy induction pathway than a retinoid. Additional optionalcomponents include a cardiotrophic inhibitor such as a CT-1 antagonist,an ACE inhibitor, such as captopril, and/or human growth hormone and/orIGF-I in the case of congestive heart failure, or with anotheranti-hypertrophic, myocardiotrophic factor, anti-arrhythmic, orinotropic factor in the case of other types of heart failure or cardiacdisorder.

The present invention can be combined with current therapeuticapproaches for treatment of heart failure, e.g., with ACE inhibitortreatment. ACE inhibitors are angiotensin-converting enzyme inhibitingdrugs which prevent the conversion of angiotensin I to angiotensin II.The ACE inhibitors may be beneficial in congestive heart failure byreducing systemic vascular resistance and relieving circulatorycongestion. ACE inhibitors include drugs designated by the trademarksAccupril® (quinapril), Altaces (ramipril), Capoten® (captopril),Lotensin® (benazepril), Monopril® (fosinopril), Prinivil® (lisinopril),Vasotec® (enalapril), and Zestril® (lisinopril).

The present invention can be combined with the adminstration of drugtherapies for the treatment of heart diseases such as hypertension. Forexample, a retinoic acid compound can be administered with endothelinreceptor antagonists, for example, an antibody to the endothelinreceptor, and peptide or other small molecule antagonists;β-adrenoceptor antagonists such as carvedilol; α₁-adrenoceptorantagonists; anti-oxidants; compounds having multiple activities (e.g.,β-blocker/α-blocker/anti-oxidant); carvedilol-like compounds orcombinations of compounds providing multiple functions found incarvedilol; growth hormone, etc.

Retinoic acid receptors RAR (α, β and γ) and RXR (α, β and γ) functionbiologically to active DNA transcription in the forms of the RXRhomodimer and RAR/RXR heterodimer. RXRα subtype may be an key targetbased on in vivo data observed with dominant, negative RXRα-mutantsubtype expressed in transgenic mice. Pan-agonists, those that havespecificity for both RAR and RXR, are preferred agonists since it is theheterodimer (RAR/RXR) that likely mediates the retinoid hypertrophysuppression effect. Less preferred are those that recognize only RAR.And least preferred are those that recognize only RXR. However, none ofthe known retinoid receptor agonists appear to have specificity solelyfor only one family; some cross-specificity can exist as evidence by theability of LG64 (RXR specific) to suppress at high concentrations. Alsopreferred are those that are specific for a RAR subtype (α, β, or γ)that mediates suppression of ventricular muscle cell hypertrophy.

Retinoid agonists alone or in combination with other hypertrophysuppressor pathway agonists or with molecules that antagonize knownhypertrophy induction pathways, are useful as drugs for in vivotreatment of mammals experiencing heart failure, so as to prevent orlessen hypertrophic effects.

Therapeutic formulations of agonist(s) for treating heart disorders areprepared for storage by mixing the agonist(s) having the desired degreeof purity with optional physiologically acceptable carriers, excipients,or stabilizers (Remington's Pharmaceutical Sciences, 16th edition, Oslo,A., Ed., 1980), in the form of lyophilized cake or aqueous solutions.Acceptable carriers, excipients, or stabilizers are non-toxic torecipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptides; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counter ions such as sodium; and/or non-ionic surfactantssuch as Tween, Pluronics, or polyethylene glycol (PEG). Theantagonist(s) are also suitably linked to one of a variety ofnonproteinaceous polymers, e.g., polyethylene glycol, polypropyleneglycol, or polyoxyalkylenes, in the manner set forth in U.S. Pat. Nos.4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337. Theamount of carrier used in a formulation may range from about 1 to 99%,preferably from about 80 to 99%, optimally between 90 and 99% by weight.

The agonist(s) to be used for in vivo administration-must be sterile.This is readily accomplished by methods known in the art, for example,by filtration through sterile filtration membranes, prior to orfollowing lyophilization and reconstitution. The agonist(s) ordinarilywill be stored in lyophilized form or in solution.

Therapeutic agonist compositions generally are placed into a containerhaving a sterile access port, for example, an intravenous solution bagor vial having a stopper pierceable by a hypodermic injection needle.

The agonist(s) administration is in a chronic fashion using, forexample, one of the following routes: injection or infusion byintravenous, intraperitoneal, intracerebral, intramuscular, intraocular,intraarterial, or intralesional routes, orally or usingsustained-release systems as noted below. Agonist(s) is administeredcontinuously by infusion or by periodic bolus injection if the clearancerate is sufficiently slow, or by administration into the blood stream orlymph. The preferred administration mode is targeted to the heart, so asto direct the molecule to the source and minimize side effects of theagonists.

Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing theprotein, which matrices are in the form of shaped articles, e.g., films,or microcapsules. Examples of sustained-release matrices includepolyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) asdescribed by Langer et al. (1981) J. Biomed. Mater. Res. 15:167-277 andLanger (1982) Chem. Tech. 12:98-105, or poly(vinyl alcohol)),polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers ofL-glutamic acid and gamma ethyl-L-glutamate (Sidman et al. (1983)Biopolymers 22:547-556), non-degradable ethylene-vinyl acetate (Langeret al. (1981) supra), degradable lactic acid-glycolic acid copolymerssuch as the Lupron Depot™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(−)-3-hydroxybutyric acid (EP 133,988).

The agonist(s) also may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization(for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-[methylmethacylate] microcapsules, respectively), in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules), or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences,supra.

While polymers such as ethylene-vinyl acetate and lactic acid-glycolicacid enable release of molecules for over 100 days, certain hydrogelsrelease molecules for shorter time periods. When encapsulated moleculesremain in the body for a long time, they may denature or aggregate as aresult of exposure to moisture at 37° C., resulting in a loss ofbiological activity and possible changes in immunogenicity. Rationalstrategies can be devised for stabilization depending on the mechanisminvolved, e.g., using appropriate additives, and developing specificpolymer matrix compositions.

Sustained-release agonist(s) compositions also include liposomallyentrapped agonist(s). Liposomes containing agonist(s) are prepared bymethods known per se: DE 3,218,121; Epstein et al. (1985) Proc. Natl.Acad. Sci. USA 82:3688-3692; Hwang et al. (1980) Proc. Natl. Acad. Sci.USA 77:4030-4034; EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP142,641; Japanese patent application 83-118008; U.S. Pat. Nos. 4,485,045and 4,544,545; and EP 102,324. Ordinarily the liposomes are of the small(about 200-800 Angstroms) unilamellar type in which the lipid content isgreater than about 30 mol % cholesterol, the selected proportion beingadjusted for the optimal agonist therapy. A specific example of asuitable sustained-release formulation is in EP 647,449.

An effective amount of agonist(s) to be employed therapeutically willdepend, for example, upon the therapeutic objectives, the route ofadministration, and the condition of the patient. Accordingly, it willbe necessary for the clinician to titer the dosage and modify the routeof administration as required to obtain the optimal therapeutic effect.A typical daily dosage of a retinoic acid compound used alone mightrange from about 1 μg/kg to up to 100 mg/kg of patient body weight ormore per day, depending on the factors mentioned above, preferably about10 μg/kg/day to 50 mg/kg/day.

If the two agonists are administered together, they need not beadministered by the same route, nor in the same formulation. However,they can be combined into one formulation as desired. Both agonists canbe administered to the patient, each in effective amounts, or each inamounts that are sub-optimal but when combined are effective. Preferablysuch amounts are about 10 μg/kg/day to 50 mg/kg/day of each. In oneembodiment, the administration of both agonists is by injection using,e.g., intravenous or subcutaneous means, depending on the type ofagonist employed. Typically, the clinician will administer theagonist(s) until a dosage is reached that achieves the desired effectfor treatment of the heart dysfunction. For example, the amount would beone which decreases ventricular muscle cell hypertrophy, increasesventricular contractility, and decreases peripheral vascular resistanceor ameliorates or treats conditions of similar importance in congestiveheart failure patients, thus obtaining the result desired in thecontinuum of equilibria between growing and shrinking of cardiac muscletissue. The progress of this therapy is easily monitored by conventionalassays.

The two types of agonists, if used together, may be formulated togetherin an appropriate carrier vehicle to form a pharmaceutical compositionthat preferably does not contain cells. In one embodiment, the bufferused for formulation will depend on whether the composition will beemployed immediately upon mixing or stored for later use, sincelong-term storage may bring into issue stability such as solubility andaggregation that can be addressed by altering the pH. The finalpreparation may be a stable liquid or lyophilized solid.

The agonist(s) optionally is combined with or administered in concertwith other agents for treating congestive heart failure, including ACEinhibitors, CT-1 inhibitors, human growth hormone, and/or IGF-I.

The effective amounts of such agents, if employed, will be at theclinician's discretion. Dosage administration and adjustment aredetermined by methods known to those skilled in the art to achieve thebest management of congestive heart failure and ideally takes intoaccount-use of diuretics or digitalis, and conditions such ashypotension and renal impairment. The dose will additionally depend onsuch factors as the type of drug used and the specific patient beingtreated. Typically the amount employed will be the same dose as thatused if the drug were to be administered without agonist; however, lowerdoses may be employed depending on such factors as the presence ofside-effects, the condition being treated, the type of patient, and thetype of agonist and drug, provided the total amount of agents providesan effective dose for the condition being treated.

Thus, for example, in the case of ACE inhibitors, a test dose ofenalapril is 5 mg, which is then increased up to 10-20 mg per day, oncea day, as the patient tolerates it. As another example, captopril isinitially administered orally to human patients in a test dose of 6.25mg and the dose is then escalated, as the patient tolerates it, to 25 mgtwice per day (BID) or three times per day (TID) and may be titrated to50 mg BID or TID. Tolerance level is estimated by determining whetherdecrease in blood pressure is accompanied by signs of hypotension. Ifindicated, the dose may be increased up to 100 mg BID or TID. Captoprilis produced for administration as the active ingredient, in combinationwith hydrochlorothiazide, and as a pH stabilized core having an entericor delayed release coating which protects captopril until it reaches thecolon. Captopril is available for administration in tablet or capsuleform. A discussion of the dosage, administration, indications andcontraindications associated with captopril and other ACE inhibitors canbe found in the Physicians Desk Reference, Medical Economics DataProduction Co., Montvale, N.J. 2314-2320 (1994).

In an example of an oral therapeutic composition of retinoic acidsuitable for treatment of ventricular muscle cell hypertrophy comprisestablets, capsules of hard or soft gelatin methylcellulose, or of anothersuitable material easily dissolved in the digestive tract. The oraldosages the formulation contains 1% retinoic acid and 99% soft gelatinmethylcellulose. In another example of an oral dosage, the formulationcontains 20 mg 9-cis retinoic acid and 70 mg gelatin. In an example ofan injectable therapeutic composition of retinoic acid, the formulationcontains 1% retinoic acid and 99% saline, wherein retinoic acid is thesodium or potassium salt thereof. In another example of an injectabletherapeutic composition of retinoic acid, the formulation contains 5% ofthe potassium salt of retinoic acid, 1% ACE inhibitor captopril, and 94%saline.

The following examples are meant to illustrate, but not to limit, theinvention.

EXAMPLE 1 Effect of Retinoic Acid on the Hypertrophic Response ofVentricular Muscle Cells.

The effects of retinoic acid on the hypertrophic response of ventricularmuscle cells to the α₁-adrenergic agonist phenylephrine (Phe) wasstudied in a cultured neonatal rat ventricular muscle cell model(Knowlton et al. (1991) J. Biol. Chem. 266:7759-7768; Shubeita et al.(1990) J. Biol. Chem. 265:20555-20562, both references hereinspecifically incorporated by reference).

Cell Culture Techniques

Neonatal rat ventricular myocytes were prepared and cultured aspreviously described (Iwaki et al. (1990) J. Biol. Chem.265:13809-13817, herein specifically incorporated by reference).Briefly, hearts from 1-2 day old Sprague-Dawley rats were recovered, andthe ventricular portions dissected and collected. Ventricular cells weredispersed by digestion with collagenase II (Worthington) and pancreatin(GIBCO). Myocytes were purified by centrifugation through Percoll stepgradients, resulting in a purification of greater than 95% myocytes (Senet al. (1988) J. Biol. Chem. 263:19132-19136), and plated at a densityof 2×10⁵ per well of the two well chamber slide precoated with 15 μg/mllaminin, or at a density of 2.5×10⁵ per well of the 24 well7 tissueculture plate precoated with 1% gelatin. The cells were culturedovernight in 4:1 Dulbecco's modified Eagles medium/medium 199 (GIBCO),supplemented with 10% fetal bovine serum (“FBS”), and antibiotics (100U/ml penicillin and 100 μg/ml streptomycin). Cells were then refed withfresh medium containing 4×10⁻⁷ M of retinoic acid or its agonists (TTNPBor 3-methyl-TTNPB) for at least 10 hours for the pretreatment, thenwashed 3 times with serum-free medium and cultured in serum-freemaintenance media (4:1 Dulbecco's modified Eagles medium/medium 199 withantibiotics) supplemented with phenylephrine (Phe), endothelin, orretinoic acid (RA) alone, or Phe or endothelin with RA for 48-72 hr.Concentrations were Phe, 2×10⁻⁵ M; endothelin, 1×10⁻⁸ M; RA, 5×10⁻⁷ M.

Immunofluorescence Techniques and Morphological Analysis.

Indirect immunofluorescence assays were performed as previouslydescribed (Iwaki et al. (1990) supra). After a 24 hr attachment, themyocytes were washed 3 times with medium without serum, then cultured inmedia as described above containing in serum-free media (maintenancemedia), Phe, Phe and all-trans retinoic acid (RA), RA alone, 10% FBS, orboth FBS and RA for 48-72 hr. The cells were rinsed with PBS, then fixedfor 15 min at room temperature with 3% paraformaldehyde in 10 mM NaPO₄,150 mM NaCl, 1 mM MgCl₂, pH 7.4. The cells were then incubated in 50 mMNH₄Cl for 10 min, washed twice with PBS, then permeabilized with 0.2%Triton X-100 in PBS for 15 min at room temperature, followed by3-additional PBS washes. The chamber slides were incubated with 1%bovine serum albumin for 10 min to block non-specific sites, incubatedwith TrpE/MLC-2v antisera (Sen et al. (1988) supra) for 60 min at 37°C., rinsed and washed 4-times with PBS. Subsequently, the chamber slideswere incubated for 60 min at 37° C. with FITC-conjugated goatanti-rabbit IgG in PBS, and then rinsed and washed 4 times with PBS. Theslides were mounted on glass coverslips with H-1000 Vectashield MountingMedium (Vecta), viewed and photographed by fluorescence microscopy. Cellimages were projected onto paper and individual cells were traced.Individual tracings were cut from the paper and weighted for estimationof cell size.

Results

Cells cultured in maintenance media showed a relatively small cell size,poorly organized myofilaments, and poor contractile ability, whereascells cultured in the presence of Phe exhibit an altered phenotype, withincreased size, well organized myofilaments, and regular contractions.In cells cultured in the presence of Phe and RA, the Phe-stimulatedincrease in cell size was largely reduced, whereas the myofilaments werestill well organized and cells continued to exhibit contractile ability.Cells cultured in the presence of RA alone showed little change in cellphenotype. The hypertrophic response is stimulated by the multiplegrowth factors present in serum. Retinoic acid had no effect onserum-treated cells.

To more precisely quantitate the effects of RA, multiple fields fromindependent cultured cell preparations were examined, utilizing apreviously described morphometric assay for the hypertrophic phenotype(Knowlton et al. (1993) J. Biol. Chem. 268:15374-15380). Results aresummarized in Table 1. Cell size was estimated by measuring the areawhich individual cells attached on the tissue culture chamber slides.Data is the means ±S.E of values from two experiments.

TABLE 1 Effect of Retinoic Acid on Ventricuiar Muscle Cell Size. NUMBER*RELATIVE MEDIUM OF CELLS CELL SIZE MAINTENANCE 23 100 ± 6  RA 1 × 10⁻⁷M 31 140 ± 14 RA 1 × 10⁻⁶ M 40 120 ± 15 Phe. 5 × 10⁻⁶ M  35 320 ± 21Phe. 5 × 10⁻⁶ M + RA 1 × 10⁻⁷ M 39 210 ± 20 Phe. 5 × 10⁻⁶ M + RA 1 ×10⁻⁶ M 31 160 ± 17 10% of FBS 26 300 ± 25 10% OF FBS + RA 1 × 10⁻⁶ M 25290 ± 34

EXAMPLE 2 Effect of Retinoic Acid on Cardiac Gene Expression.

Total RNA was isolated from primary ventricular myocytes cultured inmaintenance medium alone (control), or in the presence of 5×10⁻⁶ M Phe,5×10⁻⁶ M Phe and 1×10⁻⁶ M RA, or 1×10⁻⁶ M RA alone by the guanidine HClmethod (Chirgwin et al. (1979) Biochemistry 18:5294-5299). Northern blothybridizations were performed as described previously (Iwaki et al.(1.990) supra). 20 μg of each sample RNA was fractionated by agarose gelelectrophoresis and transferred to nylon membranes, and hybridized withradiolabeled CDNA probes of ANF (Glembotski et al. (1987) Endocrinology12:843-852), myosin light chain-2v (MLC-2v) (Kummar et al. (1986) J.Biol. Chem. 261:2866-2872), troponin I (TnI) (provided by Dr. S. Evans,University of California, San Diego), and glucose-6-phosphatasedehydrogenase (GADPH). The extent of hybridization was quantitated bydensitometry of the corresponding autoradiogram, and normalized to thesignals obtained with GADPH to correct for differences in loading andtransfer efficiencies. Results are shown in Tables 2 and 3. Cells werealso cultured in 0, 104 M, 10⁻⁷ M, or 10⁻⁶ M RA, and probed withradiolabeled ANF and GADPH cDNAs. Results are shown in Table 3.

TABLE 2 Phe and RA Effect on Cardiac RNA Expression Maintenance PhePhe + RA RA ANF 0.7 4.2 1.1 0.5 MLC-2v 0.5 0.7 1.5 0.9 TnI 2.3 1.9 2.31.7

TABLE 3 Dose-Dependency of RA Effect on Cardiac RNA Expression RA (M) 01 × 10⁻⁸ 1 × 10⁻⁷ 1 × 10⁻⁶ ANF 6.6 2.5 1.5 2.1

EXAMPLE 3 Transfection Analysis of ANF-Luciferase Fusion Gene inVentricular Muscle Cells.

To test the effect of RA on Phe stimulated ANF promoter activity, thefollowing plasmids were used in transient transfection assays:pANF(-3003)LΔ5′, the ANF/luciferase fusion gene, composed of a 3003 bpfragment of the ANF 5′-flanking region (Argentin et al. (1985) J. Biol.Chem. 260:4568-4571) which was inserted into the luciferase reportervector pSVOALγ5′ (de Wet et al. (1987) Mol. Cell. Biol. 7:725-737);pRSVLγ5′ with a RSV promoter fused with luciferase-gene, pSVOALγ5′; apromoterless vector; and pON249 a β-galactosidase expression vector witha CMV promoter (Cherrington et al. (1989) J. Virol. 63:1435-1440).

Neonatal rat ventricular myocytes were prepared and cultured asdescribed above. After 24 hr attachment of primary myocytes in 24 wellplates, cells were refed with fresh medium containing 10% FBS for 4 hr;then transfected with 0.65 μg DNA containing 500 ng of pANF(−3003)Lγ5′,50 ng of pON249, and either 50 ng of RA receptor expression vector and50 ng of inactive control plasmid pSVOALγ5′, or 100 ng of controlplasmid, using the standard calcium phosphate method (Gorman et al.(1982) Mol. Cell. Biol. 9:5022-5033). Following overnight incubation,cells were washed 3 times with PBS, then cultured with serum-freemaintenance medium (1:-RA-Phe), 2×10⁻⁶ M Phe (2:-RA+Phe), or 1×10⁻⁶ MRA+2×10⁻⁶ M Phe (3:-RA+Phe) and incubated for 48 hr. Cells were washedtwice with PBS, then lysed in 100 μl extraction buffer (100 nM KPO₄, pH7.4, 1 mM DTT, 0.5% Triton X-100) on ice for 15 min. 20 μl of eachextract was assayed in an Autolumat LB953 (Berthold) with an assaybuffer consisting of 100 mM Tricine, 10 mM MgSO₄, 2 mM EDTA, 1 mM DTT, 4mM ATP, 7.5 nM luciferin, pH 7.8. β-galactosidase activity was assayedon 10 μl samples as described previously (Rosenthal (1987) MethodsEnzymol. 152:704-720). Luciferase activities were normalized to theircorresponding β-galactosidase activities to correct for variation intransfection efficiency. Results are shown in Fig. lA. Phe induced geneexpression was suppressed to control levels in the presence of RA.

Control cells were transfected with a Rous sarcoma virus (RSV)promoter-luciferase gene construct (pRSVLγ5′) and cultured as above.Results are shown in FIG. 1B. Phe induction of gene expression wasfurther enhanced in the presence of RA.

Cells transfected with the ANF promoter-luciferase gene construct werecultured in maintenance medium, 2×10⁻⁶ M Phe, 1×10⁻⁸ M endothelin-1, or10% FBS with and without 1×10⁻⁶ M RA. Results are shown in FIG. 1C. Phe,endothelin-1, and serum induced gene expression. RA suppressed Phe andendothelin-1 gene expression to control levels; serum-induced geneexpression was not suppressed by RA.

EXAMPLE 4 Activity of RA Receptors

To assay for the activity of retinoic acid receptors in culturedventricular muscle cells, RXR-specific response element CRBP II orRAR-RXR heterodimer-specific response element β-RE I were cloned into anenhancer-dependent heterologous reporter context and introduced bytransfection as described in Example 3. The following plasmids were usedin transient transfection assays: CMV-hRARα, composed of a CMV promoterand human RARα cDNA (Umesono et al. (1991) Cell 65:1-20); CMV-hRXRαcontaining CMV promoter and human RXRα CDNA (Mangelsdorf et al. (1990)Nature 345:224-229). The luciferase reporter constructs, CRBPII-luciferase and β-RE I-luciferase, containing retinoic acid responseelements were made in a truncated TK promoter context using sequencesfrom the rat CRBP II gene (Mangelsdorf et al. (1991) Cell 66:555-561) orthe mouse gene RARβ2 gene (Sucov et al. (1990) Proc. Natl. Acad. Sci.USA 87:5392-5396) promoters.

Reporter plasmids containing CRBP II-luciferase or β-RE I-luciferasewere separately transfected into ventricular myocardial cells without orwith co-transfected expression plasmids which contain human RXRα or RARαcDNA driven by a CMV promoter. Ligands, 9-cis (9cRA) (5×10⁻⁷ M) orall-trans RA (atRA) (5×10⁻⁷ M), were separately added to the cultures toactivate CRBP II or β-RE1 promoters (Heyman et al. (1992) supra).

Results. The effects of 9cRA or atRA on the promoter activities areshown in FIG. 2A. The CRBP II reporter construct was not activated byendogenous receptors in the presence of 9cRA, but was up-regulated whenco-transfected with an RXR-expression construct. The ORE heterodimerreporter gene was functional in the presence of atRA using theendogenous complement of receptors, and this activity was furtherincreased by co-transfection of receptor expression constructs.

EXAMPLE 5 Effect of Dominant Negative RA Receptor on Gene Expression.

The reporter construct containing the ANF promoter was transfected aloneor with a dominant negative reporter construct (CMV-hRAR403), or thewild type receptor construct, CMV-hRXR, or CMV-hRAR into ventricularmyocardial cells cultured in Phe (2×10⁻⁶ M) containing medium with orwithout atRA (1×10⁻⁷ M). The dominant negative hRARα cDNA, acarboxy-terminal truncation at amino acid 403 referred to as hRARα403,has been previously described (Damm et al. (1993) Proc. Natl. Acad. Sci.USA 90:2989-2993), and is expressed from the CMV promoter.

In the presence of the dominant negative mutant receptor, RA did notsuppress Phe induction of gene expression from the ANF promoterconstruct (FIG. 2B).

EXAMPLE 6 Effect of RA Receptor-Specific Synthetic Retinoids on PheInduction of Expression.

Ventricular cells were transfected and with the ANF-luciferase reportergene and activated by 2×10⁻⁶ M Phe with a series of concentrations ofthree agonists; LG64, TTNPB, and 3-methyl-TTNPB. 9-cis retinoic acid,TTNPB, 3-methyl TTNPB, and LG64 were provided by Dr Richard Heyman(Ligand Pharmaceuticals). All compounds were diluted at least 1:1000 intissue culture media.

The effects of LG64, 3TTNPB, and TTNPB on relative luciferase activityare shown in FIG. 3A. TTNPB and 3-methyl-TTNPB are equivalent in theirpotency to block induction of the ANF promoter, whereas theRXR-selective compound LG64 is approximately 40-fold less potent in thisassay.

EXAMPLE 7 Effect of Hormones on Phe-Induced Gene Expression.

Cultured cardiomyocytes were transfected with the ANF promoter reporterconstruct and co-cultured with 2×10⁻⁷ M Phe and one of the following:dexamethasone (Dex), thyroid hormone (T3), 17.6-estradiol (E2),1,25-dihydroxy-vitamin D (D3), linoleic acid (LA), and RA (each presentin a concentration of 1×10⁻⁷ M). Phe (Sigma) was dissolved inmaintenance medium and filtered through a 0.22 μm (diameter) membrane.All-trans RA was dissolved in a small amount of DMSO, then diluted with100% EtOH. Dex, E2, and T3 (all from Sigma) were dissolved in 100% EtOH.D3 and LA were provided by Dr. Barry Forman (Salk Institute) dissolvedin EtOH. All compounds were diluted to at least 1:1000 in tissue culturemedium. As shown in FIG. 3B, only treatment with RA prevented theinduction of the ANF promoter. Treatment with thyroid hormone, estrogen,and vitamin D were inactive in this assay, and dexamethasone (asynthetic glucocorticoid) actually increased ANF expression.

EXAMPLE 8 Testing for In Vivo Suppression of Veritricular Muscle CellHypertrophy.

A. Normal Rats

A purified retinoic acid compound to be tested for suppression ofventricular muscle cell hypertrophy is administered to normal rats, andits effect on cardiovascular parameters such as blood pressure, heartrate, systemic vascular resistance, contractility, force of heart beat,concentric or dilated hypertrophy, left ventricular systolic pressure,left ventricular mean pressure, left ventricular systolic pressureend-diastolic pressure, cardiac output, stroke index,. histologicalparameters, and ventricular size and wall thickness.

B. Pressure-Overload Mouse Model

A purified retinoic acid compound is also tested in thepressure-overload mouse model wherein the pulmonary artery isconstricted, resulting in right ventricular failure.

C. RV Murine Dysfunctional Model

A retroviral murine model of ventricular dysfunction can be used to testthe ability of a purified retinoic acid compound to suppress ventricularmuscle cell hypertrophy, by assaying dP/dt, ejection fraction, andvolumes with the above-described hypertrophy assay. In this model, thepulmonary artery of the mouse is constricted so as to generate pulmonaryhypertrophy and failure.

D. Transgenic Mouse Model

Transgenic mice that harbor a muscle actin promoter-IGF-I fusion genedisplay cardiac and skeletal muscle hypertrophy, without evidence ofmyopathy or heart failure. IGF-I-gene-targeted mice display defects incardiac myogenesis (as well as skeletal), including markedly decreasedexpression of ventricular muscle contractile protein genes. Purifiedretinoic acid compounds are tested in these two models.

Additionally, genetic-based models of dilated cardiomyopathy and cardiacdysfuntion, without necrosis, can be developed in transgenic andgene-targeted mice-(MLC-ras mice; aortic banding of heterozygousIGF-I-deficient mice). Another useful animal models are the RXRα mutantmouse model (Sucov et al. (1994) Genes Dev. 8:1997-1018) andRXRα-/-embryo model (Dyson et al. (1995) Proc. Natl. Acad. Sci. (InPress)). These genetically-based animal models display importantfeatures of ventricular chamber dysmorphogenesis.

E. Post-Myocardial Infarction Rat Model

A purified retinoic acid compound is also tested in a post-myocardialinfarction rat model, which is predictive of human congestive heartfailure in producing ANF. Male Sprague-Dawley (Charles River BreedingLaboratories, Inc., eight weeks of age) are acclimated to the facilityfor at least one week before surgery. Rats are fed a pelleted rat chowand water ad libitum and housed in a light- and temperature-controlledroom.

Coronary arterial ligation

Myocardial infarction is produced by left coronary arterial ligation asdescribed by Greenen et al. (1987) J. Appl. Physiol. 93:92-96 andButtrick et al. (1991) Am. J. Physiol. 260:11473-11479. The rats areanesthetized with sodium pentobarbital (60 mg/kg, intraperitoneally),intubated via tracheotomy, and ventilated by a respirator. After aleft-sided thoracotomy, the left coronary artery is ligatedapproximately 2 mm from its origin with a 7-0 silk suture. Sham animalsundergo the same procedure except that the suture is passed under thecoronary artery and then removed. All rats are handled according to the“Position of the American Heart Association on Research Animal Use”adopted Nov. 11, 1984, by the American Heart Association. Four to sixweeks after ligation, myocardial infarction could develop into heartfailure in rats. The congestive heart failure in this model reasonablymimics congestive heart failure in most human patients.

Electrocardiograms

One week after surgery, electrocardiograms are obtained under lightmetofane anesthesia to document the development of infarcts. The ligatedrats are subgrouped according to the depth and persistence ofpathological Q waves across the precordial leads (Buttrick et al. (1991)supra; Koner et al. (1983) Am. Heart J. 51:1009-1013). This provides agross estimate of infarct size and assures that large and small infarctsare not differently distributed in the ligated rats treated with aretinoic acid compound and vehicle. Confirmation is made by preciseinfarct size measurement.

Administration of a retinoic acid compound

Four weeks after surgery, a test retinoic acid compound (5 μg/kg to 50mg/kg twice a day for 15 days) or saline vehicle is injectedsubcutaneously in both ligated rats and sham controls. Body weight ismeasured twice a week during treatment. The test retinoic acid compoundis administered in saline or water as a vehicle.

Catheterization

After 13-day treatment with the test retinoic acid compound or vehicle,rats are anesthetized with pentobarbital sodium (50 mg/kg,intraperitoneally). A catheter (PE 10 fused with PE 50) filled withheparin-saline solution (50 U/ml) is implanted into the abdominal aortathrough the right femoral artery for measurement of arterial pressureand heart rate. A second catheter (PE 50) is implanted into the rightatrium through the right jugular vein for measurement of right atrialpressure and for saline injection. For measurement of left ventricularpressures and contractility (dP/dt), a third catheter (PE 50) isimplanted into the left ventricle through the right carotid artery. Forthe measurement of cardiac output by a thermodilution method, athermistor catheter (Lyons Medical Instrument Co., Sylmar, Calif.) isinserted into the aortic arch. The catheters are exteriorized at theback of the neck with the aid of a stainless-steel wire tunneledsubcutaneously and then fixed. Following catheter implantation, all ratsare housed individually.

Hemodynamic measurements

One day after catherization, the thermistor catheter is processed in amicrocomputer system (Lyons Medical Instrument, Co.) for cardia outputdetermination, and the other three catheters are connected to a ModelCP-10 pressure transducer (Century Technology Company, Inglewood, Ca)coupled to a Grass Model 7 polygraph (Grass Instrumentation, Quincy,Mass.). Mean arterial pressure (MAP), systolic arterial pressure (SAP),heart rate (HR), right atrial pressure (RAP), left ventricular systolicpressure (LVSP), left ventricular mean pressure (LVMP), left ventricularend-diastolic pressure (LVEDP), and left ventricular maximum (dP/dt) aremeasured in conscious, unrestrained rats.

For measurement of cardiac output, 0.1 ml of isotonic saline at roomtemperature is injected as a bolus via the jugular vein catheter. Thethermodilution curve is monitored by VR-16 simultrace recorders(Honeywell Co., NY) and cardiac output (CO) is digitally obtained by themicrocomputer. Stroke volume (SV)=CO/HR; cardiac index (CI)=CO/BW;systemic vascular resistance (SVR)=MAP/CI.

After measurement of these hemodynamic parameters, 1 ml of blood iscollected through the arterial catheter. Serum is separated and storedat −70° C. for measurement of retinoic acid compound levels and otherbiochemical parameters of interest.

The rats are then anesthetized with pentobarbital sodium (60 mg/kg) andthe heart arrested in diastole with an intra-atrial injection of KCl (1M). The heart is removed, and the atria and great vessels are trimmedfrom the ventricle. The ventricle is weighed and fixed in 10% bufferedformalin.

Infarct size measurements

The right ventricular free wall is dissected from the left ventricle.The left ventricle is cut in four transverse slices from apex to base.Five micrometer sections are cut and stained with Massons' trichromestain and mounted. The endocardial and epicardial circumferences of theinfarcted and non-infarcted left ventricle are determined with aplanimeter Digital Image Analyzer. The infarcted circumference and theleft ventricular circumference of all four slices are summed separatelyfor each of the epicardial and endocardial surfaces and the sums areexpressed ass a ratio of infarcted circumference to left ventricularcircumference for each surface. These two ratios are then averaged andexpressed as a percentage for infarct size.

Statistical analysis

Results are expressed as mean±SEN. Two-way and one-way analysis ofvariance (ANOVA) is performed to assess differences in parameters amonggroups. Significant differences are then subject to post hoc analysisusing the Newman-Keuls method. P<0.05 is considered significant.

Results

The mean body weight before and after treatment with the test retinoicacid compound or vehicle is not expected to be different among thetreatment groups. It is expected that the administration of a retinoicacid compound to the ligated rats in the doses set forth above willresult in improved cardiac hypertrophy and improved cardiac function incongestive heart failure.

It would be reasonably expected that the rat data may be extrapolated tohorses, cows, humans, and other mammals, correcting for the body weightof the mammal in accordance with recognized veterinary and clinicalprocedures. Using standard protocols and procedures, the veterinarian orclinician will be able to adjust the doses, scheduling, and mode ofadministration of the retinoic acid compound to achieve maximal effectsin the desired mammal being treated. Humans are expected to respond inthis manner as well.

EXAMPLE 9 Treatment of Dilated Cardiomyopathy in Human Patients.

Intervention

Patient self-administration of a retinoic acid compound or at an initialdose of 10 5- l0 μg/kg/day is proposed. The dose would be adjusteddownward for adverse effects. If no beneficial effect and no limitingadverse effects are determined at the time of re-evaluation, the dosewould be adjusted upward. Concurrent medication doses (e.g., captoprilas an ACE inhibitor and diuretics) would be adjusted at the discretionof the care provider. After the maximum dose is administered for 8weeks, the retinoic acid compound administration is stopped, andre-evaluation performed after a similar time period off treatment (or aplacebo).

Inclusion criteria

Patients would be considered for inclusion in the study if the meet thefollowing criteria: (1) dilated cardiomyopathy (DCM), idiopathic DCM, orischemic DCM without discrete areas of akinesis/dyskineses of the leftventricle (LV) on contrast ventriculography or 2D echocardiography.Evidence for impaired systolic function to include either LVend-diastolic dimension (EDD)>3.2 cm/m² BSA or EDV>82 ml/m² on 2Dechocardiography, LV fractional shortening <28% on echocardiography, orejection fraction (by contrast ventriculography or radionuclideangiography)<0.49; (2) Symptoms: New York Heart Association class III orpeak exercise VO₂<16 ml/kg/min (adjusted for age), stable for at leastone month on digoxin, diuretics, and vasodilators (ACE inhibitors); (3)Concurrent ACE inhibitor therapy; (4) Adequate echocardiographic“windows” to permit assessment of left ventricular volume and mass; (5)Ability to self-administer a retinoic acid compound according to dosageschedule and to return reliably for follow-up assessments; (6) consentof patient and patient's primary physician to participate; and (7)absence of exclusion criteria.

Exclusion criteria

Patient would be excluded from consideration for any of the followingreasons: dilated cardiomyopathy resulting from valvular heart disease(operable or not), specific treatable etiologies (including alcohol, ifabstinence has not been attempted), or operable coronary artery disease;exercise limited by chest pain or obstructive peripheral vasculardisease; chronic obstructive lung disease; diabetes mellitus or impairedglucose tolerance; history of carpal tunnel syndrome or evidence forpositive Tinells sign on examination; history of kidney stones;symptomatic osteoarthritis; inability to consent or participate inserial bicycle ergometry with invasive hemodynamic monitoring (describedbelow); or active malignancy.

Patient assessment

(1) Major assessment point:baseline; after peak stable retinoic acidcompound dose maintained for 8 weeks; after equal period after drugdiscontinuation. It is anticipated that patients would remain in thehospital for 2-3 days at the onset of active treatment, with dailyweights and laboratory data including electrolytes, phosphorus, BUN,creatinine, and glucose. Following this, they would be monitored fordaily for 2-3 additional days by (1) physical examination, (2) symptompoint score (Kelly et al. (1990) Amer. Heart J. 119:1111), (3)laboratory data (CBC; electrolytes including Mg⁺² and Ca⁺²; BUN;creatinine; phosphorus; fasting glucose and lipid profile includingtotal cholesterol, HDL-C, LDL-C, triglycerides; liver function testssuch as AST, ALT, alkaline phosphatase, total bilirubin; total protein;albumin; uric acid; and retinoic acid compound), (4) 2D, M-mode, anddoppler echocardiography, including diastolic and systolic dimensions atthe papillary muscle level, ejection fraction estimate by areaplanimetry from apical 2-chamber and 4-chamber views, estimated systolicand diastolic volumes by Simpson's rule method, and estimated leftventricular mass, doppler assessment of mitral valve inflow profile(IVRT, peak E, peak A, deceleration time, A wave duration), andpulmonary vein flow profile (systolic flow area, diastolic flow ara, Areversal duration, and velocity), (5) rest and exercise hemodynamics andmeasured oxygen consumption, using bicycle ergometry with percutaneouslyinserted pulmonary artery and arterial catheters. Perceived exertionlevel would be scored on the Borg scale, and measurements of pulmonaryartery systolic, diastolic, and mean pressures, as well as arterialpressures and pulmonary capillary wedge pressure would be measured ateach increment of workload, along with arterial and mixed venous oxygencontent for calculating cardiac output, (6) assessment of body fat andlean body mass, as well as skeletal muscle strength and endurance.

Weekly interim assessment points would include physical examination,symptom point score, and laboratory data.

Although the invention has been described with reference to thepresently preferred embodiments, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims.

What is claimed is:
 1. A method of identifying a compound whichsuppresses ventricular muscle cell hypertrophy, comprising contactingventricular muscle cells in a liquid medium with a test compoundintroduced into the liquid medium in the presence of an inducer ofventricular muscle cell hypertrophy, and measuring the development ofventricular muscle cell hypertrophy, wherein said test compound is anRXR (retinold X receptor) or RAR (retinoic acid receptor) agonist, andwherein a reduction in development of ventricular muscle cellhypertrophy, compared to control ventricular muscle cells not contactedwith the test compound, is an indication that the compound suppressesventricular muscle cell hypertrophy.
 2. The method of claim 1 whereinsaid inducer of ventricular muscle cell hypertrophy is an α₁-adrenergicagonist.
 3. The method of claim 1 wherein said inducer of ventricularmuscle cell hypertrophy is an endothelin.
 4. The method of claim 1wherein said development of ventricular muscle cell hypertrophy ismeasured by a characteristic selected from the group consisting of anincrease in cell size, induction of expression of a genetic marker ofventricular muscle cell hypertrophy, an increase in the assembly of anindividual contractile protein, accumulation of contractile units,activation of a program of immediate early gene expression, andinduction of genes encoding contractile and embryonic proteins.
 5. Themethod of claim 4 wherein said genetic marker of ventricular muscle cellhypertrophy is atrial natriuretic factor (ANF).